DNA methyaltion requirement of chrI and chrII replication: V. cholerae has two chromosomes (chrI and chrII) whereas most bacteria have one. ChrI is the primary chromosome, as it has most of the house-keeping genes, mostly homologous to their counterparts in the E. coli chromosome, including the replication genes. However, replication of chrI and the E. coli chromososme was reported to differ in terms of the requirement of methylation. In both bacteria, the origin of replication has unusually high density of GATC sites for adenine methylation. In E. coli, methylation is important for restricting replication to only once per cell cycle but this requirement is not essential for viability. We have shown this to be the case for replication of chrI also, and the original claim that methylation is essential for chrI replication was an artefact of the experimental set up. Methylation, however, is essential for chrII replication. The chrII origin is distinct from that of chrI and is similar to some of the well-studied plasmid origins. Not all plasmid origins fire once per cell cycle but the chrII origin does, like other chromosomal origins. We found that the once-per-cell-cycle replication of chrII also depends on adenine methyaltion. For chrII, methylation was additionally required for initiator binding to the origin, an essential function in the initiation process. Although methylation is widely used to control many DNA transactions, its role in mediating initiator-origin interactions is an unprecedented finding. Transition of plasmid to chromosomal mode of replication: In all organisms, DNA replication is restricted to a specific stage of the cell cycle. Understanding of how this happens in chrII with distinct replication machinery might provide a fresh perspective on this fundamental process. With this motivation, we are studying the control of chrII replication in detail. We discovered that the chrII initiator binds site-specifically to two kinds of site: One is equivalent to repeats, common in some plasmids, called iterons. However, unlike the plasmid iterons, the chrII iterons have to be methylated before they can bind the initiator. The other kind is the 39-mers, which are unrelated in sequence to iterons. The 39-mers are the primary negative regulators of replication. They enhance interactions between iterons that inactivates the origins. The iterons in turn can also dampen the inhibitory activity of the 39-mers. How the interplay between two kinds of site might align chrII replication to the cell cycle has been modeled. The model also incorporates the fact that the origin iterons, which becomes hemimethylated after the origin firing, are kept in an inactivated state due to the binding of a hemimethylation-specific protein, SeqA, for two-thirds of the cell cycle. This does not happen to plasmid iterons because they are not methylated. The chrII control system thus has diverged considerably from the plasmid system most likely to meet the demand of cell cycle specific replication. Inter-chromosome communication in V. cholerae: We hypothesize that timely replication and segregation of the two chromosomes of V. cholerae will require communication between them, so that both the chromosomes can complete the processes before cell division. An initial indication of inter-chromosomal communication has been obtained. We have been able to find conditions where replication of one of the chromosomes could be selectively prevented. It appears that preventing chrI replication can prevent/delay chromosome II replication but the reverse is not true. The mechanism of how chrI dictates chrII replication is being investigated. Communication between replication and segregation: In eukaryotes, the preparation of segregation but not the segregation itself starts with replication. In bacteria, segregation itself follows replication initiation (coreplicational segregation) and may even depend upon the act of replication. However, the reverse process of segregation influencing replication was not known until recently. In bacteria, our knowledge of chromosome segregation primarily comes from studies in plasmids, where genes dedicated to plasmid partition (par genes) were first found. Homologues of plasmid par genes have now been identified near the origin of replication in most bacteria, including V. cholerae. Both the Vibrio chromosomes have their own par genes. We have succeeded in deleting the par genes of chrI without causing much segregation or growth defect. Rather, deletion of one the two par genes (parB) promoted replication. A similar finding has also been made in B. subtilis. The two bacteria, B. subtilis and V. cholerae, have diverged more than a billion years ago but both have retained the par genes and use them for similar purposes. Using both the bacterial and yeast two-hybrid systems we have identified the bacterial replication initiator DnaA to be the direct target of Par proteins. How the Par proteins stimulate the activity of DnaA remains to be studied. Cell size and the initiation of DNA replication in bacteria: Cell division must await completion of chromosome replication and segregation of the two sister chromosomes to opposite cell halves. In eukaryotes, DNA replication is coupled to cell growth and division through the actions of cyclin-dependent kinases and associated factors. The temporal control of cell division is largely unknown in bacteria. Recently, some of the genes involved in sensing glucose concentration in the growth media have been found to regulate cell size in E. coli and in B. subtilis. Mutations in these genes make cells smaller by about 30% but do not change their growth rates. In a collaborative study, we are determining the timing of replication initiation (initiation age) in the cell cycle of these small size mutants. The initiation age seems to depend on the bacterium in question. In B. subtilis, the age remains unchanged in the mutants, indicating that achievement of a particular cell size is not obligatory for initiation. In E. coli, however, initiation is delayed until the mutants reach the size at which initiation occurs in the wild type. The delay in initiation is compensated by an increase in replication elongation rate, allowing the replication cycle to complete on time. The initiation delay could be avoided by overproducing the initiator protein, DnaA. These results are consistent with DnaA being the rate-limiting component of replication initiation in E. coli, and that the accumulation of the initiator to a level critical for initiation depends upon growth. DnaA is also rate-limiting in B. subtilis but appears not to be controlled in a growth dependent manner. Thus, although DnaA is likely to be required for initiation in all bacteria, the mechanisms governing its supply might not to be conserved. Domain analysis of chrII initiator RctB: Towards generating Vibrio specific antimicrobial agents. Given the increasing prevalence of multi-drug resistance in pathogenic vibrios, there is a need for new targets and drugs to combat these pathogens. RctB is a chrII-specific initiator and is conserved only in the family Vibrionaceae. The protein appears ideally suited for developing anti-vibrio specific drugs. Drug design is greatly facilitated if the 3-D structural information of the target protein is known. Towards this goal we have started a systematic domain analysis of RctB. Replication initiators in general have proven refractory to structural studies because they are plastic and require for activity remodeling by chaperone proteins and/or binding to specific DNA. Initial attempts to form crystals of RctB have failed. We are attempting deletion analysis to isolate functional domains. These smaller derivatives might be more amenable for structural studies.